PROJECT SUMMARY Necrotizing enterocolitis (NEC) is a life-threatening, gastrointestinal (GI) emergency affecting 7-10% of preterm infants with mortality as high as 30-50%. Leaky gut, or intestinal barrier immaturity with elevated intestinal permeability (IP), is the proximate cause of susceptibility to NEC in preterm neonates. Early detection of leaky gut is essential to identify infants at risk for NEC to prevent and reduce disease severity. No clinical factor, routine laboratory test or biomarker alone or in combination have been described that identify preterm neonates with impaired intestinal barrier function who at increased risk for NEC. The goal of this study is to develop a rapid, non-invasive screening tool to identify ?at-risk preterm infants prior to the onset of NEC. We recently revealed rapid maturation of the intestinal barrier is strongly associated with clinical factors such as early breastmilk feeding, less antibiotic exposure and later post-menstrual age, and significantly higher abundance and metabolic activities of Clostridiales and Bifidobacterium. Although the butyrate producing Clostridiales have been implicated in strengthening the intestinal epithelial barrier and reducing intestinal inflammation, it is unknown whether butyrate and potentially other short chain fatty acids (SCFAs) are associated with neonatal intestinal barrier maturation. Our preliminary data revealed butyrate production pathways, in particular Pyruvate/Acetyl-CoA pathway are significantly more abundant in low IP subjects. In this study, we propose to validate a substantiated measure of fecal microbial and/or metabolic biomarkers combined with associated neonatal factors for a rapid, non-invasive screening test. We postulate that the level(s) of metabolic activities, particularly butyrate production and potentially other SCFAs via increased colonization of Clostridiales and Bifidobacterium, are associated with intestinal barrier maturation in preterm neonates. To address our hypothesis, we will leverage existed stool samples collected from a cohort of ~200 preterms (<33 weeks), for whom both microbiota (16S rRNA gene sequencing) and IP (urine non-metabolized sugar probes lactulose and rhamnose) are available, and propose 2 aims: 1) to determine fecal metabolic activities associated with intestinal barrier maturation in preterm neonates <33 weeks gestation; and 2) to identify and characterize butyrate-producing bacteria associated with intestinal permeability in preterm neonates during the first 7-10 days of life. At the completion of the study, we will determine the biomarkers associated with healthy intestinal barrier functions and the discriminatory scheme for accurate classification of high or low IP group. Our long-term goal is to conduct a randomize clinical trial of rationally designed intervention for NEC early prevention. Refinement of the screening tool will identify infants with aberrant leaky gut, an essential prerequisite for subsequent rational design of novel strategies such as clinical trials of probiotics to promote healthy intestinal barrier functions to reduce NEC incidence.